Gene Therapy: The Biology
Allison Amore
The Biology of Gene Therapy |
While the manipulation of genes is quite simple, the introduction of the desired gene into the human body is much more complex. Several techniques are currently being studied to determine the most effective way to introduce the desired gene into the body.
In order to get a gene of interest into the body, it must first be inserted into a vector (plasmid) or carrier for the gene. A vector is a circular piece of DNA commonly found in most viruses and some bacteria. Vectors contain restriction enzyme sites. As shown below, the plasmid is cut with a restriction enzyme (EcoRI). The restriction enzyme cuts the vector at specific points. Then, the desired DNA sequence (indicated in purple) is cut with the same restriction enzyme. The two are then combined to create a complete plasmid. The vector can be carried into the cell with any of the following techniques, explained below Figure 1.
Figure 1: Example of a plasmid (vector) used in gene therapy techniques. Image obtained from http://www.accessexcellence.org/MTC/copyright.html and Access Excellence @ the National Health Museum.
Techniques for introduction of desired genes:
The following figure displays the life cycle of a retrovirus. In gene therapy, a retrovirus is manipulated to carry the desired gene in its own viral genome. The proteins on the surface of the retrovirus (red dots in the figure below) are modified to select for the tissue or part of the body in which the desired gene should be inserted. The proteins on the outside of the retrovirus recognize receptors on the surface of cells within the body and bind. The virus then attaches to the cell, enters through reverse transcription, copies the gene of interest and incorporates it into the genome of the cell. The genes are then transcribed and translated where new viruses are made and then leave the cell to infect other cells. This process is permanent since the gene of interest has been incorporated into the DNA of the cell. Every time the cell duplicates itself via mitosis, the same gene will be incorporated in the DNA.
Figure 2: Life cycle of a retrovirus. Image used with permission of Becton, Dickinson and Company.
These types of viruses act in much the same way as retroviruses. However, one main difference is that instead of having to convert the viral RNA into double stranded DNA, the adenovirus already has a double stranded DNA genome. Researchers can simply introduce the gene of interest into the genome of the adenovirus, manipulate the surface proteins and introduce it into the patient. The adenovirus follows a similar life cycle as the retrovirus except for the reverse transcription step.
Figure 3: Life cycle of the adenovirus. In the figure above, the term vector is used to represent the genome of the virus. Image obtained from the U.S. National Library of Medicine.
The gene of interest can also be introduced via an injection of pure DNA into a specific tissue. This method does not work for most tissues and it is hard to control where the DNA goes. Direct introduction of DNA only seems to work in skeletal muscle. Furthermore, this is only a site-specific fix. For example, injection of DNA into the muscle cell will only affect the site in which it was injected, not the whole muscle. However, this technique is proving beneficial to those with muscular dystrophy.
A lipid sphere is a globular structure made out of fat that has a core in which DNA can be injected. The lipid sphere can then be injected into the host. “This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell’s membrane” (Gene Therapy).
“Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell. This delivery system tends to be less effective than other options” (Gene Therapy).
How are these techniques used?
There are several ways in which the above techniques can be used in gene therapy. These include the injection of a solution containing a mixture of retroviruses, adenoviruses, lipid spheres, or raw DNA into the site of interest. The solution injected often contains stem cells or bone marrow cells that help facilitate the integration of the carrier into the body. (For more information on stem cells, please visit the 2003 Senior Colloquium website dealing with the topic.)
Another way to introduce the gene of interest is by removing some tissue from the body and manipulating it ex vivo, outside of the body, and then inserting the tissue back into the body. This technique is more difficult because there are many steps in which something can go wrong. The tissue must be removed successfully and then it must be maintained in a culture. The tissue or cells removed are then manipulated to take up the gene of interest. Once this has occurred, the tissue must be inserted back into the patient, and the genes must function properly. There is also more of an inconvenience to the patient with this method.
Germ line gene therapy is another method that is being considered. Germ line gene therapy is the manipulation of reproductive cells so that the gene is passed on to the next generation. This approach is currently not used since it has many ethical and legal implications.
Questions or Comments: Email Dr. Verna Case
Davidson College Biology Department
Davidson College
This web page was produced as an assignment for an undergraduate course at Davidson College.
The Biology of Gene Therapy |